LETTER doi:10.1038/nature18323 Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq Barbara Treutlein1,2*, Qian Yi Lee1,3,4*, J. Gray Camp5, Moritz Mall3,4, Winston Koh1, Seyed Ali Mohammad Shariati6, Sopheak Sim3, Norma F. Neff1, Jan M. Skotheim6, Marius Wernig3,4§ & Stephen R. Quake1,7,8§ Direct lineage reprogramming represents a remarkable with Ascl1 only (hereafter referred to as Ascl1-only cells) using PCA conversion of cellular and transcriptome states1–3. However, and identified three distinct clusters (A, B, C), which correlated with the the intermediate stages through which individual cells progress level of Ascl1 expression (Fig. 1b–e). Cluster A consisted of all control during reprogramming are largely undefined. Here we use single- d0 MEFs and a small fraction of day 2 cells (~ 12%) which showed cell RNA sequencing4–7 at multiple time points to dissect direct no detectable Ascl1 expression, suggesting these day 2 cells were not reprogramming from mouse embryonic fibroblasts to induced infected with the Ascl1 virus. This is consistent with typical Ascl1 neuronal cells. By deconstructing heterogeneity at each time infection efficiencies of about 80–90%. We found that the day 0 MEFs point and ordering cells by transcriptome similarity, we find that were surprisingly homogeneous, with much of the variance due to cell the molecular reprogramming path is remarkably continuous. cycle (Extended Data Fig. 1b–g, Supplementary Data 3, Supplementary Overexpression of the proneural pioneer factor Ascl1 results in a Information). Cluster C was characterized by high expression of Ascl1, well-defined initialization, causing cells to exit the cell cycle and Ascl1-target genes (Zfp238, Hes6, Atoh8 and so on) and genes involved re-focus gene expression through distinct neural transcription in neuron remodelling, as well as the downregulation of genes involved factors. The initial transcriptional response is relatively in cell cycle and mitosis (Fig. 1c, e, f and Supplementary Data 2). homogeneous among fibroblasts, suggesting that the early steps Cluster B cells represent an intermediate population that expressed are not limiting for productive reprogramming. Instead, the Ascl1 at a low level, and were characterized by a weaker upregulation later emergence of a competing myogenic program and variable of Ascl1-target genes and less efficient downregulation of cell cycle transgene dynamics over time appear to be the major efficiency genes compared to cluster C cells. This suggests that an Ascl1 expres- limits of direct reprogramming. Moreover, a transcriptional state, sion threshold is required to productively initiate the reprogramming distinct from donor and target cell programs, is transiently induced process. In addition, we found that forced Ascl1 expression resulted in in cells undergoing productive reprogramming. Our data provide less intracellular transcriptome variance, a lower number of expressed a high-resolution approach for understanding transcriptome states genes (Fig. 1d) and a lower total number of transcripts per single cell during lineage differentiation. (Extended Data Fig. 2a, b). Notably, the distribution of average expres- Direct lineage reprogramming bypasses an induced pluripotent stage sion levels per gene was similar for all experiments independent of to directly convert somatic cell types. Using the three transcription Ascl1 overexpression (Extended Data Fig. 2c). We observed that the factors Ascl1, Brn2 and Myt1l (BAM), mouse embryonic fibroblasts upregulation of neuronal targets and downregulation of cell cycle genes (MEFs) can be directly reprogrammed to induced neuronal (iN) cells in response to Ascl1 expression are uniform, indicating that the initial within 2 to 3 weeks at an efficiency of up to 20%8. Several groups have transcriptional response to Ascl1 is relatively homogenous among all further developed this conversion using transcription factor combi- cells (Fig. 1e). This suggests that most fibroblasts are initially competent nations that almost always contain Ascl1 (refs 9–12). Recently, one of to reprogram and later events must be responsible for the moderate our groups showed that Ascl1 is an ‘on target’ pioneer factor initiating reprogramming efficiency of about 20%. the reprogramming process13, and inducing conversion of MEFs into To explore the effect of transgene copy number variation on the functional iN cells alone, albeit at a much lower efficiency compared heterogeneity of the early response, we analysed single-cell tran- to BAM14. These findings raised the question whether and when a scriptomes of an additional 47 cells induced with Ascl1 for two days heterogeneous cellular response to the reprogramming factors occurs from secondary MEFs derived via blastocyst injection from a clonal, during reprogramming and which mechanisms might cause failure Ascl1-inducible embryonic stem cell line. As expected, the induction of reprogramming. We hypothesized that single-cell RNA sequencing efficiency of Ascl1 was 100% since the secondary MEFs are genetically (RNA-seq) could be used as a high-resolution approach to reconstruct identical and all cells carry the transgene in the same genomic location the reprogramming path of MEFs to iN cells and uncover mechanisms (Fig. 1g). Nevertheless, these clonal MEFs had similar transcriptional limiting reprogramming efficiencies4,15,16. responses and heterogeneity as primary infected MEFs at the day 2 time In order to understand transcriptional states during direct conver- point, as well as comparable reprogramming efficiencies and maturation sion between somatic fates, we measured 405 single-cell transcriptomes (Extended Data Fig. 3a). Finally, we compared the early response in our (Supplementary Data 1) at multiple time points during iN cell repro- Ascl1-only single-cell RNA-seq data with our previously reported bulk gramming (Fig. 1a and Extended Data Fig. 1a). We first explored how RNA-seq data of Ascl1-only and BAM-mediated reprogramming13 individual cells respond to Ascl1 overexpression during the initial phase (Extended Data Fig. 3b). We found similar downregulation of MEF- of reprogramming. We analysed day 0 MEFs and day 2 cells induced related genes and upregulation of pro-neural marker genes in both 1Department of Bioengineering, Stanford University, Stanford, California 94305, USA. 2Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig 04103, Germany. 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California 94305, USA. 4Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA. 5Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305, USA. 6Department of Biology, Stanford University, Stanford, California 94305, USA. 7Howard Hughes Medical Institute, Stanford, California 94305 USA. 8Department of Applied Physics, Stanford University, Stanford, California 94305, USA. *These authors contributed equally to this work. §These authors jointly supervised this work. 00 MONTH 2016 | VOL 000 | NATURE | 1 © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER + − a Single-cell RNA-seq measurements b Cluster C BA 40 Tau–eGFP and 15 Tau–eGFP cells for transcriptome analysis − d0 d2 d5 d22 25 by fluorescence-activated cell sorting. We found that Tau–eGFP BAM cells lacked expression of neuronal Ascl1-target genes (genes B), and Ascl1 0 MEF Infected Clonal Tau–eGFP+ iN cell maintained expression of fibroblast-associated genes (genes A and C; + –25 Tau–eGFP– Fig. 2a, b, Extended Data Fig. 4a, Supplementary Data 4). In addition, PC2 (2.11%) d0 2 d2 we found a positive correlation (R =​ 0.49) between Ascl1 expres- c 25 d 2.0 ×10–4 A sion and Tau–eGFP intensities (Extended Data Fig. 4b, Fig. 2a, b). y Ascl1 6.0 B log 0 Quantitative real-time (qRT)–PCR and western blot analysis of Ascl1 1.5 2 C [FPKM] 5 4.0 expression on day 5 to day 12 Tau–eGFP-sorted cells validated a 1.0 − –25 0 significant decrease in Ascl1 expression in Tau–eGFP cells compared 0.5 2.0 d0 PC2 (2.11%) + d2 Single cell densit to Tau–eGFP cells (Fig. 2c, Supplementary Data 5). Thus, Ascl1 0 0 –50 –25 025 3.0 4.0 5.0 3,000 5,000 7,000 expression is correlated to Tau–eGFP levels and expression of neu- PC1 (12.58%) Transcriptome variance No. of expressed genes ronal genes at day 5. This raises the hypothesis that Ascl1 is silenced e Genes I Genes II in cells that fail to reprogram. Alternatively, cells with low or no Ascl1 Cluster expression at day 5 and day 22 might have never highly expressed A Ascl1. To distinguish between these two mechanisms, we used live cell Cluster microscopy to track cells over a time course from 3–6 days after Ascl1 B induction using an eGFP–Ascl1 fusion construct (Fig. 2d, Extended Cluster C Data Fig. 5). We immunostained the cells at day 6 using Tuj1 antibod- ies recognizing the neuronal β 3-tubulin (Tubb3) to identify cells that 1 5 3 3 d0 b 0 12 d2 Tcf4 Sox9 Snca Cav1 Hes6 Birc Cnn2 Ascl Pval log [FPKM] Tcf12 differentiated towards neuronal fate. We found that transgenic Ascl1 Atoh8 2 Foxp2 Tubb Cdca Ube2c Zfp238 Hmga2 Ppp3ca protein levels varied substantially over time and, on average, contin- f Genes I –log10[P value] Genes II –log10[P value] g Single + 0 10 0 10 cells d0 MEF ued to increase over time in Tuj1 cells, but decreased or plateaued Translation Reg. pol II transcription − y 20 d2 Ascl1 Cell division Reg. gene expression infected in Tuj1 cells, leading to a significant difference in Ascl1 expression M phase Striated muscle dev. 10 d2 Ascl1 Cell cycle Reg. exc. memb. pot. clonal within six days of Ascl1 induction (Fig. 2e, Extended Data Fig. 4c). Rnp complex Axon Densit 0 –0.10 0.1 Organelle Cell projection BP This time-lapse analysis demonstrated that Ascl1 is silenced in many Ribosome Neuron projection CC PC1 loadings cells that fail to reprogram. Figure 1 | Ascl1 overexpression elicits a homogeneous early response We next analysed the maturation events occurring during late and initiates expression of neuronal genes.